Bvh-a2 and bvh-a3 antigens of group b streptococcus

ABSTRACT

Group B  Streptococcus  polypeptides and polynucleotides encoding them are disclosed. Said polypeptides may be useful for the prophylaxis, diagnostic and/or therapy of streptococcal infection in mammals. Also disclosed are recombinant methods of producing the polypeptide antigens as well as diagnostic assays for detecting streptococcal infections, particularly GBS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/398,570, now allowed, which has a filing date of Sep. 26, 2003, andwhich is a national stage application filed under 35 U.S.C. §371 ofInternational Patent Application PCT/CA01/01465, accorded aninternational filing date of Oct. 15, 2001, which claims the benefitU.S. Provisional Application No. 60/239,919 filed Oct. 13, 2000, all ofwhich applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 484112_(—)421C1_SEQUENCE_LISTING.txt. The textfile is 29 KB, was created on Jan. 22, 2008, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

FIELD OF THE INVENTION

The present invention is related to polypeptides of Group BStreptococcus (GBS) (S. agalactiae) and corresponding DNA fragments,which may be useful to prevent, diagnose and/or treat GBS infections inindividuals such as humans.

BACKGROUND OF THE INVENTION

Streptococcus are gram (+) bacteria that are differentiated by groupspecific carbohydrate antigens A through O found on their cell surface.Streptococcus groups are further distinguished by type-specific capsularpolysaccharide antigens. Several serotypes have been identified for theGBS: Ia, Ib, II, III, IV, V, VI, VII and VII. GBS also containsantigenic proteins known as “C-Proteins” (alpha, beta, gamma and delta),some of which have been cloned.

Although GBS is a common component of the normal human vaginal andcolonic flora this pathogen has long been recognized as a major cause ofinfections in neonates, expectant mothers, some non-pregnant adults aswell as mastitis in dairy herds. Expectant mothers exposed to GBS are atrisk of postpartum infection and may transfer the infection to theirbaby as the child passes through the birth canal.

GBS infections in infants are restricted to very early infancy.Approximately 80% of infant infections occur in the first days of life,so-called early-onset disease. Late-onset infections occur in infantsbetween 1 week and 2 to 3 months of age. Clinical syndromes of GBSdisease in newborns include sepsis, meningitis, pneumonia, cellulitis,osteomyelitis, septic arthritis, endocarditis, and epiglottis. Inaddition to acute illness due to GBS, which is itself costly, GBSinfections in newborns can result in death, disability, and, in rareinstances, recurrence of infection. Although the organism is sensitiveto antibiotics, the high attack rate and rapid onset of sepsis inneonates and meningitis in infants results in high morbidity andmortality.

Among pregnant women, GBS causes clinical illness ranging from mildurinary tract infection to life-threatening sepsis and meningitis,including also osteomyelitis, endocarditis, amniotis, endometritis,wound infections (postcesarean and postepisiotomy), cellulitis,fasciitis.

Among non-pregnant adults, the clinical presentations of invasive GBSdisease most often take the form of primary bacteremia but also skin ofsoft tissue infection, pneumonia, urosepsis, endocarditis, peritonitis,meningitis, empyema. Skin of soft tissue infections include cellulitis,infected peripheral ulcers, osteomyelitis, septic arthritis and decubitior wound infections. Among people at risk, there are debilitated hostssuch as people with a chronic disease such as diabetes mellitus andcancer, or elderly people.

GBS infections can also occur in animals and cause mastitis in dairyherds.

To find a vaccine that will protect hosts from GBS infection, researcheshave turned to the type-specific antigens. Unfortunately thesepolysaccharides have proven to be poorly immunogenic in hosts and arerestricted to the particular serotype from which the polysaccharideoriginates. Further, capsular polysaccharide elicit a T cell independentresponse i.e. no IgG production. Consequently capsular polysaccharideantigens are unsuitable as a vaccine component for protection againstGBS infection.

Others have focused on the C-protein beta antigen which demonstratedimmunogenic properties in mice and rabbit models. This protein was foundto be unsuitable as a human vaccine because of its undesirable propertyof interacting with high affinity and in a non-immunogenic manner withthe Fc region of human IgA. The C-protein alpha antigen is rare in typeIII serotypes of GBS which is the serotype responsible for most GBSmediated conditions and is therefore of little use as a vaccinecomponent.

There remains an unmet need for GBS polypeptides that may be useful toprevent, diagnose and/or treat GBS infections in individuals such ashumans.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 70% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8 or fragments or analogs thereof.

According to one aspect, the present invention relates to polypeptideswhich comprise an amino acid sequence selected from SEQ ID NOS: 3, 4, 7and 8 or fragments or analogs thereof.

In other aspects, there are provided polypeptides encoded bypolynucleotides of the invention, pharmaceutical compositions, vectorscomprising polynucleotides of the invention operably linked to anexpression control region, as well as host cells transfected with saidvectors and processes of producing polypeptides comprising culturingsaid host cells under conditions suitable for expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the DNA sequence of BVH-A2 gene from serotype IIIGroup B Streptococcus strain NCS954; SEQ ID NO: 1.

FIG. 2 represents the DNA sequence of BVH-A2 gene from serotype IIIGroup B Streptococcus strain NCS954 without the region coding for itsleader peptide; SEQ ID NO: 2.

FIG. 3 represents the amino acid sequence of BVH-A2 polypeptide fromserotype III Group B Streptococcus strain NCS954; SEQ ID NO: 3.

FIG. 4 represents the amino acid sequence of BVH-A2 polypeptide fromserotype III Group B Streptococcus strain NCS954 without the 37 aminoacid residues leader peptide; SEQ ID NO: 4.

FIG. 5 represents the DNA sequence of BVH-A3 gene from serotype IIIGroup B Streptococcus strain NCS954; SEQ ID NO:5.

FIG. 6 represents the DNA sequence of BVH-A3 gene from serotype IIIGroup B Streptococcus strain NCS954 without the region coding for theleader peptide; SEQ ID NO:6.

FIG. 7 represents the amino acid sequence of BVH-A3 polypeptide fromserotype III Group B Streptococcus strain NCS954; SEQ ID NO: 7.

FIG. 8 represents the amino acid sequence of BVH-A3 polypeptide fromserotype III Group B Streptococcus strain NCS954 without the 2 aminoacid residues leader peptide; SEQ ID NO:8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides purified and isolated polynucleotides,which encode Group B Streptococcal polypeptides that may be used toprevent, treat, and/or diagnose streptococcal infection. The presentinvention provides four separate preferred polynucleotides, eachindividually and separately defined by one of SEQ ID NOS 1, 2, 5 and 6.Further provided in the present invention are four separatepolypeptides, each individually and separately defined by one of seq IDNOS: 3, 4, 7 and 8. Those skilled in the art will appreciate that theinvention includes polynucleotides that encode analogs such as mutants,variants, homologues and derivatives of such polypeptides, as describedherein in the present patent application. The invention also includesRNA molecules corresponding to the DNA molecules of the invention. Inaddition to the DNA and RNA molecules, the invention includes thecorresponding polypeptides and monospecific antibodies that specificallybind to such polypeptides.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 70% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 80% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 85% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 90% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 95% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide comprising a sequence chosen fromSEQ ID NOS: 3, 4, 7 and 8 or fragments or analogs thereof.

According to one aspect, the present invention relates topolynucleotides encoding an epitope bearing portion of a polypeptidehaving a sequence chosen from SEQ ID NOS: 3, 4, 7 and 8 or fragments oranalogs thereof.

According to one aspect, the present invention relates to epitopebearing portions of a polypeptide having a sequence chosen from SEQ IDNOS: 3, 4, 7 and 8 or fragments or analogs thereof.

According to one aspect, the present invention relates to polypeptidescomprising an amino acid sequence comprising sequences from SEQ ID NOS:3, 4, 7 and 8 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 70% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 80% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 85% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 90% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 95% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NOS: 3, 4, 7and 8.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide comprising a sequence chosen fromSEQ ID NOS: 3, 4, 7 and 8.

According to one aspect, the present invention relates topolynucleotides encoding an epitope bearing portion of a polypeptidehaving a sequence chosen from SEQ ID NOS: 3, 4, 7 and 8.

According to one aspect, the present invention relates to epitopebearing portions of a polypeptide having a sequence chosen from SEQ IDNOS: 3, 4, 7 and 8.

In a further embodiment, the present invention also relates topolynucleotides encoding a polypeptide which is able to raise antibodieshaving binding specificity for a polypeptide having a sequence chosenfrom SEQ ID NOS: 3, 4, 7 and 8 or fragments or analogs thereof.

In a further embodiment, the present invention also relates topolynucleotides encoding a polypeptide which is able to raise antibodieshaving binding specificity for a polypeptide having a sequence chosenfrom SEQ ID NOS: 3, 4, 7 and 8.

The percentage of homology is defined as the sum of the percentage ofidentity plus the percentage of similarity or conservation of amino acidtype.

One can use a program such as the CLUSTAL program to compare amino acidsequences. This program compares amino acid sequences and finds theoptimal alignment by inserting spaces in either sequence as appropriate.It is possible to calculate amino acid identity or similarity (identityplus conservation of amino acid type) for an optimal alignment. Aprogram like BLASTx will align the longest stretch of similar sequencesand assign a value to the fit. It is thus possible to obtain acomparison where several regions of similarity are found, each having adifferent score. Both types of identity analysis are contemplated in thepresent invention.

In a further embodiment, the polypeptides in accordance with the presentinvention are antigenic.

In a further embodiment, the polypeptides in accordance with the presentinvention are immunogenic.

In a further embodiment, the polypeptides in accordance with the presentinvention can elicit an immune response in an individual.

In a further embodiment, the present invention also relates topolypeptides which are able to raise antibodies having bindingspecificity to the polypeptides of the present invention as definedabove.

In a further embodiment, the present invention also relates topolypeptides which are able to raise antibodies having bindingspecificity for a polypeptide having a sequence chosen from SEQ ID NOS:3, 4, 7 and 8 or fragments or analogs thereof.

In a further embodiment, the present invention also relates topolypeptides which are able to raise antibodies having bindingspecificity for a polypeptide having a sequence chosen from SEQ ID NOS:3, 4, 7 and 8.

An antibody that “has binding specificity” is an antibody thatrecognises and binds the selected polypeptide but which does notsubstantially recognise and bind other molecules in a sample, e.g., abiological sample which naturally includes the selected peptide.Specific binding can be measured using an ELISA assay in which theselected polypeptide is used as an antigen.

In accordance with the present invention, “protection” in the biologicalstudies is defined by a significant increase in the survival curve, rateor period. Statistical analysis using the Log rank test to comparesurvival curves, and Fisher exact test to compare survival rates andnumbers of days to death, respectively, might be useful to calculate Pvalues and determine whether the difference between the two groups isstatistically significant. P values of 0.05 are regarded as notsignificant.

In an additional aspect of the invention there are providedantigenic/immunogenic fragments of the polypeptides of the invention, orof analogs thereof.

The fragments of the present invention should include one or more suchepitopic regions or be sufficiently similar to such regions to retaintheir antigenic/immunogenic properties. Thus, for fragments according tothe present invention the degree of identity is perhaps irrelevant,since they may be 100% identical to a particular part of a polypeptideor analog thereof as described herein. The present invention furtherprovides fragments having at least 10 contiguous amino acid residuesfrom the polypeptide sequences of the present invention. In oneembodiment, at least 15 contiguous amino acid residues. In oneembodiment, at least 20 contiguous amino acid residues.

The skilled person will appreciate that “fragments”, “analogs” or“derivatives” of the polypeptides of the invention will also find use inthe context of the present invention, i.e. as antigenic/immunogenicmaterial. Thus, for instance polypeptides which include one or moreadditions, deletions, substitutions or the like are encompassed by thepresent invention.

As used herein, “analogs” of the polypeptides of the invention includethose polypeptides in which one or more of the amino acid residues aresubstituted with a conserved amino acid residue (preferably conserved)and which may be natural or unnatural. In one embodiment, analogs ofpolypeptides of the invention will have about 70% identity with thosesequences illustrated in the figures or fragments thereof. That is, 70%of the residues are the same. In a further embodiment, polypeptides willhave greater than 80% identity. In a further embodiment, polypeptideswill have greater than 90% identity. In a further embodiment,polypeptides will have greater than 95% identity. In a furtherembodiment, polypeptides will have greater than 99% identity. In afurther embodiment, analogs of polypeptides of the invention will havefewer than about 20 amino acid residue substitutions, modifications ordeletions and more preferably less than 10.

In one embodiment, derivatives and analogs of polypeptides of theinvention will have about 70% homology with those sequences illustratedin the figures or fragments thereof. In a further embodiment,derivatives and analogs of polypeptides will have greater than 80%homology. In a further embodiment, derivatives and analogs ofpolypeptides will have greater than 90% homology. In a furtherembodiment, derivatives and analogs of polypeptides will have greaterthan 95% homology. In a further embodiment, derivatives and analogs ofpolypeptides will have greater than 99% homology. In a furtherembodiment, derivatives and analogs of derivatives and analogs ofpolypeptides of the invention will have fewer than about 20 amino acidresidue substitutions, modifications or deletions and more preferablyless than 10.

According to a further aspect, the invention provides polypeptideshaving at least 70% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8 or fragments oranalogs thereof.

According to a further aspect, the invention provides polypeptideshaving at least 80% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8 or fragments oranalogs thereof.

According to a further aspect, the invention provides polypeptideshaving at least 85% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8 or fragments oranalogs thereof.

According to a further aspect, the invention provides polypeptideshaving at least 90% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8 or fragments oranalogs thereof.

According to a further aspect, the invention provides polypeptideshaving at least 95% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8 or fragments oranalogs thereof.

According to a further aspect, the invention provides polypeptidescomprising a sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8 orfragments or analogs thereof.

According to a further aspect, the invention provides polypeptidescharacterized by a sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8 orfragments or analogs thereof.

According to a further aspect, the invention provides polypeptideshaving at least 70% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOs: 3, 4, 7 and 8.

According to a further aspect, the invention provides polypeptideshaving at least 80% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8.

According to a further aspect, the invention provides polypeptideshaving at least 85% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8.

According to a further aspect, the invention provides polypeptideshaving at least 90% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8.

According to a further aspect, the invention provides polypeptideshaving at least 95% identity to a second polypeptide having an aminoacid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8.

According to a further aspect, the invention provides polypeptidescomprising a sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8.

According to a further aspect, the invention provides polypeptidescharacterized by a sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8.

These substitutions are those having a minimal influence on thesecondary structure and hydropathic nature of the polypeptide. Preferredsubstitutions are those known in the art as conserved, i.e. thesubstituted residues share physical or chemical properties such ashydrophobicity, size, charge or functional groups. These includesubstitutions such as those described by Dayhoff, M. in Atlas of ProteinSequence and Structure 5, 1978 and by Argos, P. in EMBO J. 8, 779-785,1989. For example, amino acids, either natural or unnatural, belongingto one of the following groups represent conservative changes:

ala, pro, gly, gin, asn, ser, thr, val;

cys, ser, tyr, thr;

val, ile, leu, met, ala, phe;

lys, arg, orn, his;

and phe, tyr, trp, his.

The preferred substitutions also include substitutions of D-enantiomersfor the corresponding L-amino acids.

Preferably, a fragment, analog or derivative of a polypeptide of theinvention will comprise at least one antigenic region i.e. at least oneepitope.

In an alternative approach, the analogs could be fusion polypeptides,incorporating moieties which render purification easier, for example byeffectively tagging the desired polypeptide. It may be necessary toremove the “tag” or it may be the case that the fusion polypeptideitself retains sufficient antigenicity to be useful.

Thus, what is important for analogs, derivatives and fragments is thatthey possess at least a degree of the antigenicity/immunogenicity of thepolypeptides of the invention from which they are derived.

Also included are polypeptides which have fused thereto other compoundswhich alter the biological or pharmacological properties of thepolypeptide, i.e., polyethylene glycol (PEG) to increase half-life,leader or secretory amino acid sequences for ease of purification,prepro- and pro-sequences and (poly)saccharides.

Furthermore, in those situations where amino acid regions are found tobe polymorphic, it may be desirable to vary one or more particular aminoacids to more effectively mimic the different epitopes of the differentGBS strains.

Moreover, the polypeptides of the present invention can be modified byterminal —NH₂ acylation (e.g. by acetylation or thioglycolic acidamidation, terminal carboxy amidation, e.g. with ammonia or methylamine)to provide stability, increased hydrophobicity for linking or binding toa support or other molecule.

Also contemplated are hetero and homo polypeptide multimers of thepolypeptide fragments and analogues. These polymeric forms include, forexample, one or more polypeptides that have been cross-linked withcross-linkers such asavidin/biotin, glutaraldehyde ordimethylsuperimidate. Such polymeric forms also include polypeptidescontaining two or more tandem or inverted contiguous sequences, producedfrom multicistronic mRNAs generated by recombinant DNA technology.

In a further embodiment, the present invention also relates to chimericpolypeptides which comprise one or more polypeptides or fragments oranalogs or derivatives thereof as defined in the figures of the presentapplication.

In a further embodiment, the present invention also relates to chimericpolypeptides comprising two or more polypeptides having a sequencechosen from SEQ ID NOS: 3, 4, 7 and 8 or fragments or analogs thereof;provided that the polypeptides are linked as to formed a chimericpolypeptide.

In a further embodiment, the present invention also relates to chimericpolypeptides comprising two or more polypeptides having a sequencechosen from SEQ ID NOS: 3, 4, 7 and 8 provided that the polypeptides arelinked as to formed a chimeric polypeptide.

In order to achieve the formation of antigenic polymers (i.e. syntheticmultimers), polypeptides may be utilized having bishaloacetyl groups,nitroarylhalides, or the like, where the reagents being specific forthio groups. Therefore, the link between two mercapto groups of thedifferent peptides may be a single bound or may be composed of a linkinggroup of at least two, typically at least four and not more than 16, butusually not more than about 14 carbon atoms.

In a particular embodiment, polypeptide fragments or analogs of theinvention do not contain a methionine (Met) starting residue.Preferably, polypeptides will not incorporate a leader or secretorysequence (signal sequence). The signal portion of a polypeptide of theinvention may be determined according to established molecularbiological techniques. In general, the polypeptide of interest may beisolated from GBS culture and subsequently sequenced to determine theinitial residue of the mature protein and therefore the sequence of themature polypeptide.

According to another aspect of the invention, there are also provided(i) a composition of matter containing a polypeptide of the invention,together with a carrier, diluent or adjuvant; (ii) a pharmaceuticalcomposition comprising a polypeptide of the invention and a carrier,diluent or adjuvant; (iii) a vaccine comprising a polypeptide of theinvention and a carrier, diluent or adjuvant; (iv) a method for inducingan immune response against GBS, in an individual, by administering tothe individual, an immunogenically effective amount of a polypeptide ofthe invention to elicit an immune response, e.g., a protective immuneresponse to GBS; and particularly, (v) a method for preventing and/ortreating a GBS infection, by administering a prophylactic or therapeuticamount of a polypeptide of the invention to an individual in need.

Before immunization, the polypeptides of the invention can also becoupled or conjugated to carrier proteins such as tetanus toxin,diphtheria toxin, hepatitis B virus surface antigen, poliomyelitis virusVP1 antigen or any other viral or bacterial toxin or antigen or anysuitable proteins to stimulate the development of a stronger immuneresponse. This coupling or conjugation can be done chemically orgenetically. A more detailed description of peptide-carrier conjugationis available in Van Regenmortel, M. H. V., Briand J. P., Muller S.,Plaué S., <<Synthetic Polypeptides as antigens>> in LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 19 (ed.) Burdou,R. H. & Van Knippenberg P. H.(1988), Elsevier New York.

According to another aspect, there are provided pharmaceuticalcompositions comprising one or more GBS polypeptides of the invention ina mixture with a pharmaceutically acceptable carrier diluent oradjuvant. Suitable adjuvants include (1) oil in-water emulsionformulations such as MF59™, SAF™, Ribi™; (2) Freund's complete orincomplete adjuvant; (3) salts i.e. AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄)₂,Al(OH)₃, AlPO₄, silica, kaolin; (4) saponin derivatives such asStimulon™ or particles generated therefrom such as ISCOMs(immunostimulating complexes); (5) cytokines such as interleukins,interferons, macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF); (6) other substances such as carbonpolynucleotides i.e. poly IC and polyAU, detoxified cholera toxin (CTB)and E. coli heat labile toxin for induction of mucosal immunity. A moredetailed description of adjuvant is available in a review by M.Z.I Khanet al. in Pharmaceutical Research, vol. 11, No. 1 (1994) pp2-11, andalso in another review by Gupta et al., in Vaccine, Vol. 13, No. 14, pp1263-1276 (1995) and in WO 99/24578. Preferred adjuvants include QuilA™,QS21™, Alhydrogel™ and Adjuphos™.

Pharmaceutical compositions of the invention may be administeredparenterally by injection, rapid infusion, nasopharyngeal absorption,dermoabsorption, or buccal or oral.

Pharmaceutical compositions of the invention are used for the treatmentor prophylaxis of streptococcal infection and/or diseases and symptomsmediated by streptococcal infection, in particular Group A Streptococcus(S. pyogenes), Group B Streptococcus (GBS or S. agalactiae), S.dysgalactiae, S. uberis, S. nocardia as well as Staphylococcus aureus.General information about Streptococcus, and more particularly GBS, isavailable in Manual of Clinical Microbiology by P. R. Murray et al.(1995, 6^(th) Edition, ASM Press, Washington, D.C.).

In one embodiment, pharmaceutical compositions of the invention are usedfor the treatment or prophylaxis of GBS infection and/or diseases andsymptoms mediated by GBS infection.

In a particular embodiment, pharmaceutical compositions of the inventionare administered to those individuals at risk of GBS infection such aspregnant women for mild urinary tract infection to life-threateningsepsis and meningitis, including also osteomyelitis, endocarditis,amniotis, endometritis, wound infections (postcesarean andpostepisiotomy), cellulitis, fasciitis.

In a particular embodiment, pharmaceutical compositions of the inventionare administered to those individuals at risk of GBS infection such asneonates and infants for sepsis, meningitis, pneumonia, cellulitis,osteomyelitis, septic arthritis, endocarditis, epiglottis.

In a particular embodiment, pharmaceutical compositions of the inventionare administered to those individuals at risk of GBS infection such asnon-pregnant adults, for primary bacteremia but also skin of soft tissueinfection, pneumonia, urosepsis, endocarditis, peritonitis, meningitis,empyema. Skin of soft tissue infections include cellulitis, infectedperipheral ulcers, osteomyelitis, septic arthritis and decubiti or woundinfections. Among people at risk, there are debilitated individuals suchas people with a chronic disease such as diabetes mellitus and cancer,or elderly people.

In a particular embodiment, pharmaceutical compositions of the inventionare administered to those individuals at risk of GBS infection such ascattle for the treatment of mastitis in cattle.

In a further aspect, the invention provides the use of pharmaceuticalcomposition of the invention for the prophylactic or therapeutictreatment of GBS bacterial infection in an individual susceptible to GBSinfection comprising administering to said individual a therapeutic orprophylactic amount of a composition of the invention.

According to a further aspect, the GBS polypeptides of the invention maybe used in a kit comprising the polypeptides of the invention fordetection of diagnosis of GBS infection.

As used in the present application, the term “individual” includemammals. In a further embodiment, the mammals are humans. In a furtherembodiment, the mammals are non-humans, such as herds.

In a particular embodiment, pharmaceutical compositions of the inventionare administered to those individuals at risk of GBS infection such asneonates.

Pharmaceutical compositions of the invention are preferably in unitdosage form of about 0.001 to 100 μg/kg (antigen/body weight) and morepreferably 0.01 to 10 μg/kg and most preferably 0.1 to 1 μg/kg, 1 to 3times with an interval of about 1 to 6 week intervals betweenimmunisations.

Pharmaceutical compositions are preferably in unit dosage form of about0.1 μg to 10 mg and more preferably 1 μg to 1 mg and most preferably 10to 100 μg 1 to 3 times with an interval of about 1 to 6 week intervalsbetween immunizations.

In one embodiment, polynucleotides are those illustrated in SEQ ID NOS:1, 2, 5 and 6 which may include the open reading frames (ORF), encodingthe polypeptides of the invention.

In one embodiment, polynucleotides are those illustrated in SEQ ID NOS:1, 2, 5 and 6 encoding the polypeptides of the invention.

It will be appreciated that the polynucleotide sequences illustrated inthe figures may be altered with degenerated codons yet still encode thepolypeptides of the invention. Accordingly the present invention furtherprovides polynucleotides herein above described (or the complementsequence thereof) having 50% identity between sequences. In oneembodiment, at least 70% identity between sequences. In one embodiment,at least 75% identity between sequences. In one embodiment, at least 80%identity between sequences. In one embodiment, at least 85% identitybetween sequences. In one embodiment, at least 90% identity betweensequences. In a further embodiment, polynucleotides are hybridizableunder stringent conditions, i.e. having at least 95% identity. In afurther embodiment, more than 97% identity.

Suitable stringent conditions for hybridization can be readilydetermined by one of skilled in the art (see for example Sambrook etal., (1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed, ColdSpring Harbor, N.Y.; Current Protocols in Molecular Biology, (1999)Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., N.Y.).

In a further embodiment, the present invention provides polynucleotidesthat hybridize under stringent conditions to either

(a) a polynucleotide encoding a polypeptide or

(b) the complement of a polynucleotide encoding a polypeptide;

wherein said polypeptide comprises SEQ ID NO: 3, 4, 7 and 8, orfragments or analogs thereof.

In a further embodiment, the present invention provides polynucleotidesthat hybridize under stringent conditions to either

(a) a polynucleotide encoding a polypeptide or

(b) the complement of a polynucleotide encoding a polypeptide;

wherein said polypeptide comprises at least 10 contiguous amino acidresidues from a polypeptide comprising SEQ ID NO: 3, 4, 7 and 8 orfragments or analogs thereof.

In a further embodiment, the present invention provides polynucleotidesthat hybridize under stringent conditions to either

(a) a polynucleotide encoding a polypeptide or

(b) the complement of a polynucleotide encoding a polypeptide;

wherein said polypeptide comprises SEQ ID NO: 3, 4, 7 and 8.

In a further embodiment, the present invention provides polynucleotidesthat hybridize under stringent conditions to either

(a) a polynucleotide encoding a polypeptide or

(b) the complement of a polynucleotide encoding a polypeptide;

wherein said polypeptide comprises at least 10 contiguous amino acidresidues from a polypeptide comprising SEQ ID NO: 3, 4, 7 and 8.

As will be readily appreciated by one skilled in the art,polynucleotides include both DNA and RNA.

The present invention also includes polynucleotides complementary to thepolynucleotides described in the present application.

In a further aspect, polynucleotides encoding polypeptides of theinvention, or fragments or analogs thereof, may be used in a DNAimmunization method. That is, they can be incorporated into a vectorwhich is replicable and expressible upon injection thereby producing theantigenic polypeptide in vivo. For example polynucleotides may beincorporated into a plasmid vector under the control of the CMV promoterwhich is functional in eukaryotic cells. Preferably, the vector isinjected intramuscularly.

According to another aspect, there is provided a process or method ofmanufacturing for producing polypeptides of the invention by recombinanttechniques by expressing a polynucleotide encoding said polypeptide in ahost cell and recovering the expressed polypeptide product.Alternatively, the polypeptides can be produced according to establishedsynthetic chemical techniques, i.e. solution phase or solid phasesynthesis of oligopeptides which are ligated to produce the fullpolypeptide (block ligation).

General methods for obtention and evaluation of polynucleotides andpolypeptides are described in the following references: Sambrook et al.,(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed, Cold SpringHarbor, N.Y.; Current Protocols in Molecular Biology, (1999) Edited byAusubel F. M. et al., John Wiley & Sons, Inc., N.Y.; PCR CloningProtocols, from Molecular Cloning to Genetic Engineering, (1997) Editedby White B. A., Humana Press, Totowa, N.J., 490 pages; ProteinPurification, Principles and Practices, (1993) Scopes R. K.,Springer-Verlag, N.Y., 3^(rd) Edition, 380 pages; Current Protocols inImmunology, (1999) Edited by Coligan J. E. et al., John Wiley & SonsInc., N.Y., are herein incorporated by reference.

For recombinant production, host cells are transfected with vectorswhich encode the polypeptides, and then cultured in a nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes. Suitable vectors are those thatare viable and replicable in the chosen host and include chromosomal,non-chromosomal and synthetic DNA sequences e.g. bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA. The polypeptide sequence may beincorporated in the vector at the appropriate site using restrictionenzymes such that it is operably linked to an expression control regioncomprising a promoter, ribosome binding site (consensus region orShine-Dalgarno sequence), and optionally an operator (control element).One can select individual components of the expression control regionthat are appropriate for a given host and vector according toestablished molecular biology principles (Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor,N.Y.; Current Protocols in Molecular Biology, (1999) Edited by AusubelF. M. et al., John Wiley & Sons, Inc., N.Y., incorporated herein byreference). Suitable promoters include but are not limited to LTR orSV40 promoter, E. coli lac, tac or trp promoters and the phage lambdaP_(L) promoter. Vectors will preferably incorporate an origin ofreplication as well as selection markers, i.e. antibiotic resistancegene. Suitable bacterial vectors include pET, pQE70, pQE60, pQE-9, pD10phagescript, PSIX174, pBluescript SK, pbsks, pNH8A, pNH16A, pNH46A,ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic vectorspBlueBacIII, pWLNEO, pSV2CAT, pOG44, PXTI, pSG, pSVK3, pBPV, pMSG andpSVL. Host cells may be bacterial (i.e. E. coli, Bacillus subtilis,Streptomyces), fungal (i.e. Aspergillus niger, Aspergillus nidulins),yeast (i.e. Saccharomyces) or eukaryotic (i.e. CHO, COS).

Upon expression of the polypeptide in culture, cells are typicallyharvested by centrifugation then disrupted by physical or chemical means(if the expressed polypeptide is not secreted into the media) and theresulting crude extract retained to isolate the polypeptide of interest.Purification of the polypeptide from culture media or lysate may beachieved by established techniques depending on the properties of thepolypeptide using ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography hydrophobic interaction chromatography, hydroxylapatitechromatography and lectin chromatography. Final purification may beachieved using HPLC.

The polypeptide may be expressed with or without a leader or secretionsequence. In the former case, the leader may be removed usingpost-translational processing (see U.S. Pat. No. 4,431,739, U.S. Pat.No. 4,425,437 and U.S. Pat. No. 4,338,397 incorporated herein byreference) or be chemically removed subsequent to purifying theexpressed polypeptide.

According to a further aspect, the GBS polypeptides of the invention maybe used in a diagnostic test for GBS infection, in particular for GBSinfection. Several diagnostic methods are possible, for exampledetecting GBS organism in a biological sample, the following proceduremay be followed:

a. obtaining a biological sample from an individual;

b. incubating an antibody or fragment thereof reactive with an GBSpolypeptide of the invention with the biological sample to form amixture, and

c. detecting specifically bound antibody or bound fragment in themixture which indicates the presence of GBS.

Alternatively, a method for the detection of antibody specific to a GBSantigen in a biological sample containing or suspected of containingsaid antibody may be performed as follows:

a. obtaining a biological sample from an individual;

b. incubating one or more GBS polypeptides of the invention or fragmentsthereof with the biological sample to form a mixture; and

c. detecting specifically bound antigen or bound fragment in the mixturewhich indicates the presence of antibody specific to GBS.

One of skill in the art will recognize that this diagnostic test maytake several forms, including an immunological test such as anenzyme-linked immunoadsorbent assay (ELISA), a radioimmunoassay or alatex agglutination assay, essentially to determine whether antibodiesspecific for the polypeptide are present in an organism.

The polynucleotides encoding polypeptides of the invention may also beused to design DNA probes for use in detecting the presence of GBS in abiological sample suspected of containing such bacteria. The detectionmethod of this invention comprises:

a. obtaining the biological sample from an individual;

b. incubating one or more DNA probes having a DNA sequence encoding apolypeptide of the invention or fragments thereof with the biologicalsample to form a mixture; and

c. detecting specifically bound DNA probe in the mixture which indicatesthe presence of GBS bacteria.

The DNA probes of this invention may also be used for detectingcirculating GBS (i.e. GBS nucleic acids) in a sample, for example usinga polymerase chain reaction, as a method of diagnosing GBS infections.The probe may be synthesized using conventional techniques and may beimmobilized on a solid phase or may be labelled with a detectable label.A preferred DNA probe for this application is an oligomer having asequence complementary to at least about 6 contiguous nucleotides of theGBS polypeptides of the invention.

Another diagnostic method for the detection of GBS in an individualcomprises:

a. labelling an antibody reactive with a polypeptide of the invention orfragment thereof with a detectable label;

b. administering the labelled antibody or labelled fragment to theindividual; and

c. detecting specifically bound labelled antibody or labelled fragmentin the individual which indicates the presence of GBS.

A further aspect of the invention is the use of the GBS polypeptides ofthe invention as immunogens for the production of specific antibodiesfor the diagnosis and in particular the treatment of GBS infection.Suitable antibodies may be determined using appropriate screeningmethods, for example by measuring the ability of a particular antibodyto passively protect against GBS infection in a test model. One exampleof an animal model is the mouse model described in the example herein.The antibody may be a whole antibody or an antigen-binding fragmentthereof and may belong to any immunoglobulin class. The antibody orfragment may be of animal origin, specifically of mammalian origin andmore specifically of murine, rat or human origin. It may be a naturalantibody or a fragment thereof, or if desired, a recombinant antibody orantibody fragment. The term recombinant antibody or antibody fragmentmeans antibody or antibody fragment which was produced using molecularbiology techniques. The antibody or antibody fragments may be polyclonalor preferably monoclonal. It may be specific for a number of epitopesassociated with the GBS polypeptides but is preferably specific for one.

A further aspect of the invention is the use of a pharmaceuticalcomposition of the invention for the prophylactic or therapeutictreatment of GBS infection comprising administering to said individual aprophylactic or therapeutic amount of the composition.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belong. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLE 1

This example illustrates the identification of GBS BVH-A2 and BVH-A3genes.

Chromosomal DNA was isolated from different GBS strains as previouslydescribed (Jayarao B M et al. 1991. J. Clin. Microbiol.29: 2774-2778). AλZAPExpress genomic library was constructed using chromosomal DNApurified from the serotype III GBS strain NCS954 and screened accordingto the manufacturer's instruction (STRATAGENE, La Jolla, Calif.) with apool of human normal sera. Briefly, the purified chromosomal DNA waspartially digested with tsp509I restriction enzyme, and the resultingfragments were electrophoresed on a 1% agarose gel (Bio-Rad). Fragmentsin the 5-to 10-kb size range were extracted from the gel and ligated tothe EcoRI arms of λZAPExpress vector and the vector was encapsidatedusing the Gigapack II packaging extract (STRATAGENE). The recombinantphages were used to infect E. coli XLI-Blue MRF′[Δ(mcrA)183Δ(mcrCB-hsdSMR-mrr)173 endAl supE44 thi-1 recA1 gyrA96 relAllac (F′ proAB lacl^(q)ZΔM15 Tn10 [Tet^(R)])], which was then plated ontoLB agar. The resulting plaques were lifted onto Hybond-C nitrocellulosemembranes (AMERSHAM PHARMACIA Biotech, Baie d'Urfée, Canada)pre-impregnated with 10 mM Isopropyl-β-d-thiogalactopyranoside (IPTG:ICN Biomedicals Inc., Costa Mesa, Calif.). The membranes were blockedusing phosphate-buffered saline (PBS) with 3% skim milk and weresequentially incubated with the pooled of human sera, peroxydase-labeledgoat anti-human immunoglobulins antisera (Jackson ImmunoresearchLaboratories Inc., West Grove, Pa.) and substrate. Positive plaques wereisolated, purified twice, and the recombinant pBK-CMV plasmids(STRATAGENE) were excised with the ExAssist helper phage (STRATAGENE)according to the manufacturer's instructions. Immunoblots using phagemidvectors containing the cloned inserts revealed that the pooled humansera reacted with a protein band with an approximate molecular weight of65 kDa for the clone H31-29, while it reacted with two protein bandswith an approximate molecular weights between 40-60 kDa for the cloneF8. These clones were respectively identified as BVH-A2 and BVH-A3. Thesequence of the inserts were determined using the TAQ Dye DeoxyTerminator Cycle Sequencing Kit with an APPLIED BIOSYSTEMS Inc. (FosterCity, Calif.) automated sequencer model 373A according to themanufacturer's recommendations.

EXAMPLE 2

This example illustrates the cloning of GBS BVH-A2 and BVH-A3 genes.

The coding regions of Group B streptococcal BVH-A2 (SEQ ID NO: 1) andBVH-A3 (SEQ ID NO: 5) genes were respectively amplified by PCR (DNAThermal Cycler GeneAmp PCR system 2400 Perkin Elmer, San Jose, Calif.)from purified recombinant phagemid clone H31-29 and genomic DNA ofserotype III Group B streptococcal strain NCS954 using oligonucleotideprimers that contained base extensions for the addition of restrictionsites Ndel (CATATG) and Xhol (CTCGAG). The oligonucleotide primers(Table 1) DMAR172 (SEQ ID NO:9) and DMAR173 (SEQ ID NO:10) were used toamplify the BVH-A2 gene, while DMAR204 (SEQ ID NO:15) and DMAR205 (SEQID NO:16) were used to amplify the BVH-A3 gene. PCR products werepurified from agarose gel using a QIAQUICK gel extraction kit fromQIAGEN following the manufacturer's instructions (Chatsworth, Calif.),and digested with Ndel and Xhol (Pharmacia Canada Inc, Baie d'Urfé,Canada). The pET-21b(+) vector (NOVAGEN, Madison, Wis.) was digestedwith Ndel and Xhol and purified from agarose gel using a QIAQUICK gelextraction kit from QIAGEN (Chatsworth, Calif.). The Ndel-Xhol PCRproducts were ligated to the Ndel-Xhol pET-21b(+) expression vector. Theligated products were transformed into E. coli strain DH5α [φ80dlacZΔM15Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (r_(K)−m_(K)+) deoR thi-1 supE44λ⁻gyrA96 relA1] (GIBCO BRL, Gaithersburg, Md.) according to the methodof Simanis (Hanahan, D. DNA Cloning, 1985, D. M. Glover (ed), pp.109-135). Recombinant pET-21b (+)plasmids (rpET21b(+)) containing BVH-A2or BVH-A3 genes were purified using a QIAGEN plasmid kit (Chatsworth,Calif.) and DNA inserts were sequenced (Taq Dye Deoxy Terminator CycleSequencing kit, ABI, Foster City, Calif.).

It was determined that the open reading frame (ORF) which codes forBVH-A2 gene (SEQ ID NO: 1) contains 1626-bp and encodes a 541 amino acidresidues polypeptide with a predicted pl of 8.99 and a predictedmolecular mass of 59730.66 Da. Analysis of the predicted amino acidresidues sequence (SEQ ID NO:3) using the Spscan software (WisconsinSequence Analysis Package; Genetics Computer Group) suggested theexistence of a 37 amino acid residues signal peptide(MRGSLSTKQSYSLRKYKFGLASVILGSFIMVTSPVFA) (SEQ ID NO:22), which end with acleavage site situated between an alanine and a aspartic acid residues.Analysis of this ORF did not revealed the presence of repetitivestructures, or IgA binding motif (MLKKIE) (SEQ ID NO:21), but a putativecell wall anchoring motif (LPKTG) (SEQ ID NO:23) was identified near theC-terminal end between amino acid residues 479 and 483. Comparison ofthe amino acid sequence of BVH-A2 (SEQ ID NO.3) with the sequencescompiled in the available databanks revealed 18% identity with anhypothetical 40 kDa transmembrane exported protein of Streptococcusmutans which was located upstream the sr gene encoding the SR proteinimplicated in the interactions of S. mutans with salivary glycoproteins(GeneBank accession number: c60328: Ogier et al 1991. Infection andImmunity 59:1620-1626).

TABLE 1 Oligonucleotide primers used for PCR amplifications of CBSBVH-A2 and BVH-A3 genes Oligonucleotides Genes primers I.D. SequencesBVH-A2 DMAR172 5′-CTTTGGGGAACATATGAGGGGATC (SEQ ID NO:9) TC-3′ BVH-A2DMAR173 5′-CTAAAAAGATTTACTCGAGAATTT (SEQ ID NO:10) CAATATAGCG-3′ BVH-A2DMAR373 5′-ATGAGGGGATCTCTCAGTACTAAG (SEQ ID NO:11) CAATCTT-3′ BVH-A2DMAR374 5′-TTAAATTTCAATATAGCGACGAAT (SEQ ID NO:12) ACCGGA-3′ BVH-A2DMAR464 5′-CATAGGATCCGGATCAAACTACAT (SEQ ID No 13) CGGTTCAAG-3′ BVH-A2DMAR465 5′-CCGGGTCGACTTAAATTTCAATAT (SEQ ID NO:14) AGCGACG-3′ BVH-A3DMAR204 5′-CACAGGAGAACATATGAAGATTAA (SEQ ID NO:15)AAAAATTATTAGTGGCTTTGCC-3′ BVH-A3 DMAR205 5′-CTTTCTCGAGTGCACCTTGATGGC(SEQ ID NO:16) GATCAGC-3′ BVH-A3 DMAR466 5′-CATAGGATCCTGATGACACCACCA(SEQ ID NO:17) GTGAGTATCACTATATC-3′ BVH-A3 DMAR4675′-CATAGTCGACTTATGCACCTTGAT (SEQ ID NO:18) GGCGATCAG-3′

It was determined that the open reading frame (ORF) which codes forBVH-A3 gene (SEQ ID NO:5) contains 1590-bp and encodes a 529 amino acidresidues polypeptide with a predicted pl of 6.14 and a predictedmolecular mass of 59019.48 Da. Analysis of the predicted amino acidresidues sequence (SEQ ID NO:7) using the Spscan software (WisconsinSequence Analysis Package; Genetics Computer Group) suggested theexistence of a 28 amino acid residues signal peptide(MKIKKIISGFAAALIISSLSTINYEVKA) (SEQ ID NO:19), which ends with acleavage site situated between an alanine and an aspartic acid residues.Analysis of this ORF did not revealed the presence of repetitivestructures, cell wall anchoring motif (LPXTG) (SEQ ID NO:20), or IgAbinding motif (MLKKIE) (SEQ ID NO:21). Comparison of the amino acidsequence of BVH-A3 (SEQ ID NO:7) with the sequences compiled in theavailable databanks did not reveal any significant homology withsequences available in the databanks.

EXAMPLE 3

This example describes the PCR amplification of GBS BVH-A2 and BVH-A3genes from other GBS strains

To confirm the presence by PCR amplification of BVH-A2 (SEQ ID NO:1) andBVH-A3 (SEQ ID NO:5) genes, the following 11 serologically distinct GBSstrains were used: C388/90 (serotype Ia/c), ATCC12401 (serotype Ib),ATCC27591 (serotype Ic), NCS246 (serotype II/R), NCS954 (serotype III),NCS97SR331 (serotype IV), NCS535 (serotype V), NCS9842 (serotype VI),NCS7271 (serotype VII), NCS970886 (serotype VIII), ATCC27956 (bovineisolate). These strains were obtained from the American Type CultureCollection (Rockville, Md., USA) and National Centre for Streptococcus,Provincial Laboratory of Public Health for Northern Alberta (Edmonton,Canada). The E. coli strain XL1-Blue MRF′ was used in these experimentsas negative control. Chromosomal DNA was isolated from each Group Bstreptococcal strain as previously described (Jayarao B M et al. 1991.J. Clin. Microbiol. 29:2774-2778). BVH-A2 (SEQ ID NO:1) and BVH-A3 (SEQID NO:5) genes were amplified by PCR (DNA Thermal Cycler GeneAmp PCRsystem 2400 Perkin Elmer, San Jose, Calif.) from the genomic DNApurified from the 11 GBS strains, and the control E. coli strain usingthe oligonucleotides presented in Table 1. The oligonucleotide primersDMAR373 (SEQ ID NO:11) and DMAR374 (SEQ ID NO:12) were used to amplifythe BVH-A2 (SEQ ID NO:1) gene, while DMAR204 (SEQ ID NO:15) and DMAR205(SEQ ID No:16) were used to amplify the BVH-A3 (SEQ ID NO:5) gene. PCRwas performed with 35 cycles of 45 sec at 94° C., 45 sec at 55° C. and 2min at 72° C. and a final elongation period of 10 min at 72° C. The PCRproducts were size fractionated in 1% agarose gels and were visualizedby ethidium bromide staining. The results of these PCR amplificationsare presented in Table 2. The analysis of the amplification productsrevealed that both BVH-A2 (SEQ ID NO:1) and BVH-A3 (SEQ ID NO:5) geneswere present in the genome of all of the 11 GBS strains tested. No suchproduct was detected when the control E. coli DNA was submitted toidentical PCR amplifications with both sets of oligonucleotide primers.

TABLE 2 Identification of BVH-A2 and BVH-A3 genes by PCR amplificationIdentification by PCR Strains identification amplification of GBSisolates BVH-A2 BVH-A3 C388/90 (serotype Ia/c) + + ATCC12401 (serotypeIb) + + ATCC27591 (serotype Ic) + + NCS246 (serotype II/R) + + NCS954(serotype III) + + NCS97SR331 (serotype IV) + + NCS535 (serotype V) + +NCS9842 (serotype VI) + + NCS7271 (serotype VII) + + NCS970886 (serotypeVIII) + + ATCC27956 (bovine isolate) + + E. coli control strain XL1 BlueMRF' − −

EXAMPLE 4

This example illustrates the cloning of GBS BVH-A2 and BVH-A3 genes inCMV plasmid pCMV-GH.

The DNA coding region of Group B streptococcal BHV-A2 (SEQ ID NO:4) andBVH-A3 (SEQ ID NO:8) polypeptides were inserted in phase downstream of ahuman growth hormone (hGH) gene which was under the transcriptionalcontrol of the cytomegalovirus (CMV) promoter in the plasmid vectorpCMV-GH (Tang et al., Nature, 1992,356:152). The CMV promotor is nonfunctional plasmid in E. coli cells but active upon administration ofthe plasmid in eukaryotic cells. The vector also incorporated theampicillin resistance gene.

The coding regions of BVH-A2 (SEQ ID NO: 2) and BVH-A3 (SEQ ID NO: 6)genes without their leader peptide regions were amplified by PCR (DNAThermal Cycler GeneAmp PCR system 2400 Perkin Elmer, San Jose, Calif.)from genomic DNA of serotype III GBS strain NCS954 using oligonucleotideprimers that contained base extensions for the addition of restrictionsites BamHI (GGATCC) and SalI (GTCGAC). The oligonucletide primersDMAR464 (SEQ ID NO:13) and DMAR465 (SEQ ID NO:14) were used to amplifythe BVH-A2 (SEQ ID NO:2) gene, while DMAR466 (SEQ ID NO:17) and DMAR467(SEQ ID NO:18) were used to amplify the BVH-A3 (SEQ ID NO:6) genes. ThePCR products were purified from agarose gel using a QIAQUICK gelextraction kit from QIAGEN (Chatsworth, Calif.), digested withrestriction enzymes (Pharmacia Canada Inc, Baie d'Urfe, Canada). ThepCMV-GH vector (Laboratory of Dr. Stephen A. Johnston, Department ofBiochemistry, The University of Texas, Dallas, Tex.) was digested withBamHI and SalI and purified from agarose gel using the QIAQUICK gelextraction kit from QIAGEN (Chatsworth, Calif.). The BamHI-SalI DNAfragments were ligated to the BamHI-SalI pCMV-GH vector to create thehGH-BVH-A2 and hGH BVH-A3 fusion polypeptides under the control of theCMV promoter. The ligated products were transformed into E. coli strainDH5α [φ80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17 (r_(K)−m_(K)+)deoR thi-1 supE44 λ⁻gyrA96 relA1] (GIBCO BRL, Gaithersburg, Md.)according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D. M.Glover (ed), pp. 109-135). The recombinant pCMV plasmids were purifiedusing a QIAGEN plasmid kit (Chatsworth, Calif.) and the nucleotidesequences of the DNA inserts were verified by DNA sequencing.

EXAMPLE 5

This example illustrates the use of DNA to elicit an immune response toGBS BVH-A2 and BVH-A3 polypeptide antigens.

Groups of 8 female BALB/c mice (Charles River, St-Constant, Québec,Canada) are immunized by intramuscular injection of 100 μl three timesat two- or three-week intervals with 50 μg of recombinant pCMV-GHencoding BVH-A2 (SEQ ID NO:2) or BVH-A3 (SEQ ID NO:6) genes in presenceof 50 μg of granulocyte-macrophage colony-stimulating factor(GM-CSF)-expressing plasmid pCMV-GH-GM-CSF (Laboratory of Dr. Stephen A.Johnston, Department of Biochemistry, The University of Texas, Dallas,Tex.). As control, groups of mice are injected with 50 μg of pCMV-GH inpresence of 50 μg of pCMV-GH-GM-CSF. Blood samples are collected fromthe orbital sinus prior to each immunization and seven days followingthe third injection and serum antibody responses are determined by ELISAusing either purified BVH-A2-His·Tag or BVH-A3-His·Tag recombinantpolypeptides as coating antigens.

EXAMPLE 6

This example illustrates the production and purification of recombinantGBS BVH-A2 and BVH-A3 polypeptides.

The recombinant pET-21b(+)plasmids with BVH-A2 or BVH-A3 genesrespectively corresponding to the SEQ ID NO: 1, and SEQ ID NO: 5 areused to transform by electroporation (Gene Pulser II apparatus, BIO-RADLabs, Mississauga, Canada) E. coli strain BL21 (DE3) (F^(−ompT hsdS)_(B) (r⁻ _(B)m⁻ _(B)) gal dcm (DE3)) (NOVAGEN, Madison, Wis.). In thisstrain of E. coli, the T7 promotor controlling expression of therecombinant polypeptide is specifically recognized by the T7 RNApolymerase (present on the λDE3 prophage) whose gene is under thecontrol of the lac promotor which is inducible byisopropyl-β-d-thio-galactopyranoside (IPTG). The transformants BL21(DE3)/rpET are grown at 37° C. with agitation at 250 rpm in LB broth(peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) containing 100 μg ofcarbenicillin (SIGMA-ALDRICH Canada Ltd., Oakville, Canada) per mL untilthe A₆₀₀ reaches a value of 0.6. In order to induce the production ofGBS BVH-A2-His·Tag and BVH-A3-His·Tag recombinant polypeptides, thecells are incubated for 3 additional hours in the presence of IPTG at afinal concentration of 1 mM. Induced cells from a 500 ml culture arepelleted by centrifugation and frozen at −70° C.

The purification of the recombinant polypeptides from the solublecytoplasmic fraction of IPTG-induced BL21 (DE3)/rpET21b(+) is done byaffinity chromatography based on the properties of the His·Tag sequence(6 consecutive histidine residues) to bind to divalent cations (Ni²⁺)immobilized on the His·Bind metal chelation resin. Briefly, the pelletedcells obtained from a 500 mL culture induced with IPTG are resuspendedin lysis buffer (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9)containing 1 mM PMSF, sonicated and centrifuged at 12,000×g for 20 minto remove debris. The supernatant is deposited on a Ni—NTA agarosecolumn (QIAGEN, Mississauga, Ontario, Canada). The GBS BVH-A2-His·Tagand BVH-A3-His·Tag recombinant polypeptides are eluted with 250 mMimidazole-500 mM NaCl-20 mM Tris pH 7.9. The removal of the salt andimidazole from the samples is done by dialysis against PBS at 40° C. Thequantities of recombinant polypeptides obtained from the solublefraction of E. coli are estimated by MicroBCA® (Pierce, Rockford, Ill.).

EXAMPLE 7

This example illustrates the accessibility to antibodies of the GBSBVH-A2 and BVH-A3 polypeptides at the surface of GBS strains.

Bacteria are grown in Todd Hewitt (T H) broth (DIFCO Laboratories,Detroit Mich.) with 0.5% Yeast extract (DIFCO Laboratories) and 0.5%peptone extract (MERCK, Darmstadt, Germany) at 37° C. in a 8% CO₂atmosphere to give an OD_(490 nm) of 0.600 (˜10⁸ CFU/ml). Dilutions ofanti-BVH-A2, anti-BVH-A3 or control sera are then added and allowed tobind to the cells, which are incubated for 2 h at 4° C. Samples arewashed 4 times in blocking buffer [phosphate-buffered saline (PBS)containing 2% bovine serum albumin (BSA)], and then 1 mL of goatfluorescein (FITC)-conjugated anti-mouse IgG+IgM diluted in blockingbuffer is added. After an additional incubation of 60 min at roomtemperature, samples are washed 4 times in blocking buffer and fixedwith 0.25% formaldehyde in PBS buffer for 18-24 h at 4° C. Cells arewashed 2 times in PBS buffer and resuspended in 500 μl of PBS buffer.Cells are kept in the dark at 4° C. until analyzed by flow cytometry(Epics® XL; BECKMAN COULTER, Inc.).

EXAMPLE 8

This example illustrates the protection against fatal GBS infectioninduced by passive immunization of mice with rabbit hyper-immune sera.

New Zealand rabbits (Charles River laboratories, St-Constant, Canada)are injected subcutaneously at multiple sites with 50 μg and 100 μg ofBVH-A2-His·Tag or BVH-A3-His·Tag polypeptides that are produced andpurified as described in Example 6 and adsorbed to ALHYDROGEL adjuvant(SUPERFOS Biosector a/s). Rabbits are immunized three times atthree-week intervals with the BVH-A2-His·Tag or BVH-A3-His·Tagpolypeptides. Blood samples are collected three weeks after the thirdinjection. The antibodies present in the serum are purified byprecipitation using 40% saturated ammonium sulfate. Groups of 10 femaleCD-1 mice (Charles River) are injected intravenously with 500 μl ofpurified serum collected either from BVH-A2-His·Tag, or BVH-A3-His·Tagimmunized rabbits, or rabbits immunized with an unrelated controlrecombinant protein. Eighteen hours later the mice are challenged withapproximately 8×10⁴ CFU of the GBS strain C388/90 (Ia/c). Samples of theGBS challenge inoculum are plated on blood agar plates to determine theCFU and to verify the challenge dose. Deaths are recorded for a periodof 14 days.

EXAMPLE 9

This example illustrates the protection of mice against fatal GBSinfection induced by immunization.

Groups of 8 female CD-1 mice (Charles River) are immunizedsubcutaneously three times at three-week intervals with 20 μg of eitherBVH-A2-His·Tag or BVH-A3-His·Tag polypeptides that are produced andpurified as described in Example 6 in presence of 10 μg of QuilA™adjuvant (Cedarlane Laboratories Ltd, Hornby, Canada). The control miceare injected with QuilA™ adjuvant alone in PBS. Blood samples arecollected from the orbital sinus on day 1, 22 and 43 prior to eachimmunization and seven days (day 50) following the third injection. Twoweeks later the mice are challenged with approximately 8×10⁴ CPU of theGBS strain C388/90 (Ia/c). Samples of the GBS challenge inoculum areplated on blood agar plates to determine the CFU and to verify thechallenge dose. Deaths are recorded for a period of 14 days.

1. An isolated polynucleotide comprising a polynucleotide chosen from;(a) a polynucleotide encoding a polypeptide having at least 70% identityto a second polypeptide comprising a sequence chosen from: SEQ ID NOS:3, 4, 7 and 8 or fragments or analogs thereof; (b) a polynucleotideencoding a polypeptide having at least 95% identity to a secondpolypeptide comprising a sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8or fragments or analogs thereof; (c) a polynucleotide encoding apolypeptide comprising a sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8or fragments or analogs thereof; (d) a polynucleotide encoding apolypeptide which is able to raise antibodies having binding specificityfor a polypeptide having a sequence chosen from SEQ ID NOS: 3, 4, 7 and8 or fragments or analogs thereof; (e) a polynucleotide encoding anepitope bearing portion of a polypeptide having a sequence chosen fromSEQ ID NOS: 3, 4, 7 and 8 or fragments or analogs thereof; (f) apolynucleotide comprising a nucleotide sequence chosen from SEQ ID NOS:1, 2, 5 and 6 or fragments or analogs thereof; and (g) a polynucleotidecomplementary to the polynucleotide of (a), (b), (c), (d), (e) or (f).2. An isolated polynucleotide comprising a polynucleotide chosen from;(a) a polynucleotide encoding a polypeptide having at least 70% identityto a second polypeptide comprising a sequence chosen from: SEQ ID NOS:3, 4, 7 and 8; (b) a polynucleotide encoding a polypeptide having atleast 95% identity to a second polypeptide comprising a sequence chosenfrom: SEQ ID NOS: 3, 4, 7 and 8; (c) a polynucleotide encoding apolypeptide comprising a sequence chosen from: SEQ ID NOS: 3, 4, 7 and8; (d) a polynucleotide encoding a polypeptide which is able to raiseantibodies having binding specificity for a polypeptide having asequence chosen from SEQ ID NOS: 3, 4, 7 and 8; (e) a polynucleotideencoding an epitope bearing portion of a polypeptide having a sequencechosen from SEQ ID NOS: 3, 4, 7 and 8; (f) a polynucleotide comprising anucleotide sequence chosen from SEQ ID NOS: 1, 2, 5 and 6; and (g) apolynucleotide complementary to the polynucleotide of (a), (b), (c),(d), (e), or (f).
 3. The polynucleotide of claim 1, wherein saidpolynucleotide is DNA.
 4. The polynucleotide of claim 2, wherein saidpolynucleotide is DNA.
 5. The polynucleotide of claim 1, wherein saidpolynucleotide is RNA.
 6. The polynucleotide of claim 2, wherein saidpolynucleotide is RNA.
 7. The polynucleotide of claim 1 that hybridizesunder stringent conditions to either (a) a polynucleotide encoding apolypeptide or (b) the complement of a polynucleotide encoding apolypeptide; wherein said polypeptide comprises SEQ ID NO: 3, 4, 7 and 8or fragments or analogs thereof.
 8. The polynucleotide of claim 2 thathybridizes under stringent conditions to either (a) a polynucleotideencoding a polypeptide or (b) the complement of a polynucleotideencoding a polypeptide; wherein said polypeptide comprises SEQ ID NO: 3,4, 7 or
 8. 9. The polynucleotide of claim 1 that hybridizes understringent conditions to either (a) a polynucleotide encoding apolypeptide or (b) the complement of a polynucleotide encoding apolypeptide; wherein said polypeptide comprises at least 10 contiguousamino acid residues from a polypeptide comprising SEQ ID NO: 3, 4, 7 and8 or fragments or analogs thereof.
 10. The polynucleotide of claim 2that hybridizes under stringent conditions to either (a) apolynucleotide encoding a polypeptide or (b) the complement of apolynucleotide encoding a polypeptide; wherein said polypeptidecomprises at least 10 contiguous amino acid residues from a polypeptidecomprising SEQ ID NO: 3, 4, 7 or
 8. 11. A vector comprising thepolynucleotide of claim 1, wherein said polynucleotide is operablylinked to an expression control region.
 12. A vector comprising thepolynucleotide of claim 2, wherein said polynucleotide is operablylinked to an expression control region.
 13. A host cell transfected withthe vector of claim
 11. 14. A host cell transfected with the vector ofclaim
 12. 15. A process for producing a polypeptide comprising culturingthe host cell according to claim 13 under conditions suitable forexpression of said polypeptide.
 16. A process for producing apolypeptide comprising culturing the host cell according to claim 14under condition suitable for expression of said polypeptide.
 17. Anisolated polypeptide comprising a polypeptide chosen from: (a) apolypeptide having at least 70% identity to a second polypeptide havingan amino acid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8 orfragments or analogs thereof; (b) a polypeptide having at least 95%identity to a second polypeptide having an amino acid sequence chosenfrom: SEQ ID NOS: 3, 4, 7 and 8 or fragments or analogs thereof; (c) apolypeptide comprising a sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8or fragments or analogs thereof; (d) a polypeptide which is able toraise antibodies having binding specificity for a polypeptide having asequence chosen from SEQ ID NOS: 3, 4, 7 and 8 or fragments or analogsthereof; (e) an epitope bearing portion of a polypeptide having asequence chosen from SEQ ID NOS: 3, 4, 7 and 8 or fragments or analogsthereof; (f) the polypeptide of (a), (b), (c), (d) or (e) wherein theN-terminal Met residue is deleted; and (g) the polypeptide of (a), (b),(c), (d) or (e) wherein the secretory amino acid sequence is deleted.18. An isolated polypeptide comprising a polypeptide chosen from: (a) apolypeptide having at least 70% identity to a second polypeptide havingan amino acid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8; (b) apolypeptide having at least 95% identity to a second polypeptide havingan amino acid sequence chosen from: SEQ ID NOS: 3, 4, 7 and 8; (c) apolypeptide comprising a sequence chosen from: SEQ ID NOS: 3, 4, 7 and8; (d) a polypeptide which is able to raise antibodies having bindingspecificity for a polypeptide having a sequence chosen from SEQ ID NOS:3, 4, 7 and 8; (e) an epitope bearing portion of a polypeptide having asequence chosen from SEQ ID NOS: 3, 4, 7 and 8; (f) the polypeptide of(a), (b), (c), (d) or (e) wherein the N-terminal Met residue is deleted;and (g) the polypeptide of (a), (b), (c), (d) or (e) wherein thesecretory amino acid sequence is deleted.
 19. A chimeric polypeptidecomprising two or more polypeptides having a sequence chosen from SEQ IDNOS: 3, 4, 7 and 8 or fragments or analogs thereof; provided that thepolypeptides are linked as to form a chimeric polypeptide. 20.(canceled)
 21. A pharmaceutical composition comprising the polypeptideaccording to any one of claims 17 to 19 and a pharmaceuticallyacceptable carrier, diluent or adjuvant.
 22. A method for prophylacticor therapeutic treatment of group B Streptococcus (GBS) bacterialinfection in an individual susceptible to GBS infection comprisingadministering to said individual a therapeutic or prophylactic amount ofthe composition according to claim
 21. 23. A method according to claim22 wherein the individual is a neonate or an infant.
 24. A methodaccording to claim 23 wherein said infection causes sepsis, meningitis,pneumonia, cellulitis, osteomyelitis, septic arthritis, endocarditis orepiglottis.
 25. A method according to claim 22 wherein the individual isa pregnant woman.
 26. A method according to claim 25 wherein saidinfection causes mild urinary tract infection, sepsis, meningitis,osteomyelitis, endocarditis, amniotis, endometritis, postcesarean woundinfection, postepisiotomy, wound infection, cellulitis, or fasciitis.27. A method according to claim 22 wherein the individual is anon-pregnant adult.
 28. A method according to claim 27 wherein saidinfection causes primary bacteremia, skin of soft tissue infection,pneumonia, urosepsis, endocarditis, peritonitis, meningitis or empyema.29. A method according to claim 22 wherein the individual is a cow. 30.A method according to claim 29 wherein said infection causes mastitis.31. A method for diagnostic of group B Streptococcus (GBS) bacterialinfection in an individual susceptible to GBS infection comprising: a.obtaining a biological sample from an individual; b. incubating anantibody or antigen-binding fragment thereof reactive with thepolypeptide of claim 17 with the biological sample to form a mixture,and c. detecting specifically bound antibody or bound antigen-bindingfragment in the mixture which indicates the presence of GBS. 32.(canceled)
 33. (canceled)
 34. Kit comprising the polypeptide accordingto any one of claims 17 to 19 for detection or diagnosis of GBSinfection.